Anal. Chem. 1998, 70, 1357-1361
Characterization of a Capillary Zone Electrophoresis/Electrospray-Mass Spectrometry Interface Christoph Siethoff,† Walter Nigge, and Michael Linscheid*
ISAS Institute of Spectrochemistry and Applied Spectroscopy, P.O. Box 101352, D-44013 Dortmund, Germany
A dependable and stable CZE/ESI-MS interface has been constructed. To avoid instabilities in both, the capillary electrophoretic separation and the electrospray, the second of the three concentric capillaries in the three-layered sprayer has been replaced by an aluminum-coated fusedsilica capillary with an inner diameter only slightly greater than the outer diameter of the separation capillary. By this means, the otherwise often observed destruction of the separation capillary (“electrodrilling”) can be avoided completely due to the suppression of electrochemical processes leading to gas bubble formation at the tip of the sprayer. With some examples taken from different biochemical areas and by separation of natural compounds, the capability and the reliability of the modified sprayer as the central part of the interface are demonstrated. Interfacing liquid chromatography to mass spectrometry (LC/ MS), particularly to electrospray MS, is already a routine technique. The success of this combination has been overwhelming in many areas of analytical applications. Most technical problems have been solved during the last years, and complete systems are commercially available. With capillary electrophoresis (CE), the second promising separation technique, which reached some maturity in recent time, the situation has been different. Due to technical constraints, the interface still needs improvement1 and complete systems are only slowly appearing on the market. This means that a potential user has to buy the two main components separately, and the interface must be custom-made or needs at least technical optimization.2 Since 1987, the first experiments by Olivares et al.3 demonstrated that MS can be the most powerful detector not only for HPLC but for CE (generally capillary zone electrophoresis, CZE) as well; many technical improvements4,5 and applications (e.g. refs 6 and 7) have been published (for a review, see ref 8). But from † Present address: Institute of Physiological Chemistry I, University Bochum, Universita¨tsstr. 150, D-44780 Bochum, Germany. (1) Tomlinson, A. J.; Guzman, N. A.; Naylor, S. J. Capillary Electrophor. 1995, 2, 247-266. (2) Tomlinson, A. J.; Benson, L. M.; Naylor, S. J. Capillary Electrophor. 1994, 1, 127-135. (3) Olivares, J. A.; Nguyen, N. T.; Yonker, C. R.; Smith, R. D. Anal. Chem. 1987, 59, 1230. (4) Ramsey, R. S.; McLuckey, S. A. J. Microcolumn Sep. 1995, 7, 461-469. (5) Svers, J. C.; Harms, A. C.; Smith, R. D. Rapid Commun. Mass Spectrom. 1996, 10, 1175-1178.
S0003-2700(97)00950-5 CCC: $15.00 Published on Web 02/14/1998
© 1998 American Chemical Society
our own experiences,9 it became clear that some technical details have to be carefully optimized to make this interface routine or a part of an automated analytical tool. In this article, we describe our solution to this problem, which has been developed with the aim to have a robust, dependable, and automated system, which should be transferable to all electrospray ion sources. This means the system should be designed to run unattended for a extended time. Several typical applications demonstrate the capabilities of the CE/ESI-MS interface. EXPERIMENTAL SECTION Capillary Electrophoresis. All reported CE experiments were performed with the instrument PRINCE (bai, Bensheim, Germany) using a 70 cm × 50 µm i.d. fused-silica capillary column (SGE, Weiterstadt, Germany). The programmable injector allows electrokinetic sample introduction as well as hydrodynamic injection with variable pressure ranging from 0 to 2500 mbar and variable time; the resolution is 1 mbar and 0.01 min, respectively, allowing the required fine-tuning of the procedures. During the experiments described here, sample introduction was carried out hydrodynamically for 6 s in most experiments and 12 s for CID experiments applying 100-mbar injection pressure. The buffer was ammonium acetate (30 mM, pH 9.0) always. The electrophoretic separation was carried out with an effective separation voltage of 30 kV in the negative ion mode and with 22 kV in the positive ion mode, yielding a current of approximately 16-22 µA. Mass Spectrometry. Electrospray mass spectra were acquired using a MAT 90 (Finnigan MAT, Bremen, Germany) equipped with an ESI II electrospray ion source. The acceleration voltage was ∼5 kV. The electrospray operates with a voltage of (3.5 kV. For all experiments, the temperature of the heated desolvation capillary was held at 250 °C. The source and most interface parameters were tuned at the beginning of the experiments and kept unaltered during each part of this study. A makeup solution consisting of 2-propanol and electrophoretic (6) Beyerbach, A.; Farmer, P. B.; Sabbioni, G. Biomarkers 1996, 1, 9-20. (7) Wycherley, D.; Rose, M. E.; Giles, K.; Jarvis, S. A.; McDowall, M. A. Biomed. Chromatogr. 1995, 9, 283-284. (8) Cai, J.; Henion, J. J. Chromatogr., A 1995, 703, 667-692. (9) (a) Schrader, W.; Linscheid, M. Arch. Toxicol. 1997, 71, 588-595. (b) Schrader, W.; Linscheid, M. J. Chromatogr., A 1995, 717, 117-125. (c) Janning, P.; Schrader, W.; Linscheid, M. Rapid Commun. Mass Spectrom. 1994, 8, 1035-1040.
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buffer (10%) was delivered using a Harvard syringe pump (Harvard Apparatus, South Natick, MA) in a coaxial flow. Acquisition of mass spectra and selected-ion monitoring was done in the profile mode with a mass resolution of 1000. The scan rate was set to a scan cycle of ∼1 s. Chemicals. A 0.1 mg/mL aqueous solution of the dinucleotide d(TpT) (Boehringer Mannhein, Germany) was used. The antibiotic erythromycin was purchased from Sigma-Aldrich Chemie GmbH (Deisenhofen, Germany). The two peptides FYQLAKTCPV and FYQLAKTCRV, a gift from Max Planck Institute of Molecular Physiology (Dortmund, Germany), were dissolved in water to a final concentration of ∼1 mg/mL. For quantification experiments, a stock solution of five phenol carbonic acids with a concentration of 0.1 mg/mL of each compound was prepared. Solvents were high-purity grade methanol, 2-propanol (Merck, Darmstadt, Germany), and water was purified in-house with a Seralpur deltaUV system (Seral Reinstwasser Systeme, RansbachBaumbach, Germany). In negative ion mode, the electron scavenger gas SF6 (Messer-Griesheim) was used to suppress electrical discharge. CE fused-silica capillaries with 50-µm inner and 210-µm outer diameters and the aluminum-coated fused-silica with 320-µm inner and 430-µm outer diameters were obtained from SGE (Weiterstadt, Germany). RESULTS Interface Design for CZE/ESI-MS. To conserve the flexibility of the interface, the sprayer assembly was only slightly modified. The coaxial-type interface (delivered with the instruments) with three capillaries was used here, with the separation fused-silica capillary as the innermost capillary, and no further junction was built-in. The second capillary, normally made from stainless steel, carries the makeup flow and the electrospray potential. The outermost capillary allows addition of a gas flow either as a nebulizing gas such as nitrogen or an electron scavenger gas such as sulfur hexafluoride to suppress discharge especially in the negative ion mode.10 The durability of thin-wall electrophoresis capillaries in combination with the stainless steel capillary is too short for practical use due to electrodrilling,11 which destroys the capillary and interrupts the analysis. On the other hand, capillaries with small dimensions have advantages for electrospray. The thinner wall improves the wetability with makeup liquid, and the stability of the electrospray is improved. To solve this problem, we have replaced the stainless steel capillary and used aluminum-coated fused silica of similar diameters instead. The aluminum-coated fused-silica capillary (outer diameter 430 µm, inner diameter 320 µm) was cut to the same length as the original stainless steel capillary, and the sprayer end was polished until a smooth surface became visible. Sealing of the capillary was achieved with a cylindrical piece made from Delrin replacing the original version. The dimensions allow the use of separation capillaries up to 280-µm outer diameter. In the reported experiments, fused-silica capillaries of 210-µm outer diameter and 50µm inner diameter were used throughout. (10) Ikonomu, M. G.; Blades, A. T.; Kebarle, P. J. Am. Soc. Mass Spectrom. 1991, 2, 497-505. (11) Moseley, M. A.; Deterding, L. J.; Tomer, K. B.; Jorgenson, J. W. Anal. Chem. 1991, 63, 109-114.
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Figure 1. (Top) Surface of the aluminum-coated fused-silica tip (A) before and (B) after 5 h of electrospray. (Bottom) Signal area of TpT as a function of the sheath flow rate.
The sputter process and the electrochemistry of aluminum in basic solutions result in the limited usable lifetime of the spray capillaries. The surface of the capillary before and after 5 h of electrospray is shown in Figure 1. The surface is rough; the aluminum is oxidized or removed, resulting in an unstable spray, which means that regularly after approximately 5-6 h of uninterrupted work the capillary has to be replaced. But the exchange of the capillary needs a few minutes only, and the interface parameters remain unchanged. In addition, the capillary is not expensive; thus we do not consider this a severe limitation. For optimization of the sheath liquid flow rate, samples of 12 nL of 0.1 mg/mL solution of the dinucleotide d(TpT) in water were used with selected-ion monitoring; the measurements between 10 and 0.8 µL/min were repeated three times. No effect of 2-propanol itself on the signal intensity was observed. At flow rates below 1 µL/min, the spray becomes unstable. Thus, for all measurements, a flow rate of 1.2-2 µL/min was chosen because the spray conditions are still stable with good sensitivity. As an additional benefit, the reduction of sheath liquid flow yields a better signal-to-noise ratio, since the noise level is reduced and the intensity of the signal is increased (Figure 2); thus lower detection limits are achievable. Automation. One example shall demonstrate the possibility of automation of the CZE/ESI-MS analysis using the interface described above. The electropherogram in Figure 3 was obtained by programming the analysis cycle five times. A washing step with separation buffer was included in the cycles (1 min, 1000 mbar). The electrospray voltage was not turned off during the whole analysis time of 1 h. One has to realize that this results in an electrokinetic injection which adds to the normally applied hydrodynamic injection (0.1 bar, 6 s). A failure of the electrospray
Figure 2. CZE/ESI-MS mass electropherograms of d(TpT) showing the difference in the signal-to-noise ratio and the peak width dependent on a sheath flow rate of (A) 1 and (B) 5 µL/min: m/z 545, scan range m/z 500-600; injection, 0.1 bar, 12 s, 0.1 mg/mL d(TpT); 70 cm × 50 µm fused silica; 28 kV separation voltage; buffer, 30 mM ammonium acetate, pH 9.0.
Figure 3. Electropherogram of five automated injections of d(TpT). See text for conditions.
because of a sucking effect was not observed. The calculated areas of the peaks are given, and a relative standard deviation of ∼10% was obtained. Applications. Capillary electrophoresis is often applied for the analysis of peptide and protein mixtures since it allows rapid separations and low sample consumption; in addition, it provides an independent means to HPLC for the analysis of mixtures. For a demonstration of separation and structure verification in the same experiment using in-source fragmentation with CZE/ESIMS, we have chosen a mixture of two closely related peptides, the dinucleotide d(TpT) and erythromycin A; finally, the CZE/ ESI-MS analysis of a mixture of phenol carbonic acids will be discussed. The electropherogram in Figure 4 shows the separation of the two peptides FYQLAKTCPV and FYQLAKTCRV, which differ in the exchange of proline against arginine only. The mass spectra were aquired in positive ion mode scanning from m/z 100 to 1400. For CID, the tube lens voltage was set to 150 V. The mass spectra show the daughter ions, which provide information about the sequence of both peptides. The fragmentation parameters were
not optimized, but it is evident that structural information can be obtained under such conditions. The dinucleotide d(TpT) was chosen as example for negative ion fragmentation. The mass spectrum (see inset of Figure 5) has only a few chemically relevant negatively charged fragment ions which generally allow structure elucidation of oligonucleotides.12 Erythromycin, the main compound of the mixture of Streptomyces erythreus producing macrolide antibiotics, was measured in positive ion mode under standard separation conditions, since it is used often to evaluate the performance of an LC/MS interface. The in-source fragmentation with 100-V voltage at the tube lens yields two major fragments at m/z 576.5 and 158 (Figure 6). Finally, the linearity and reproducibility in quantification was investigated using a mixture of five phenolcarbonic acids. The structures of these compounds are shown in Figure 7 together with the electropherograms of the [M - H]- for all the compounds. Selected ions were monitored to allow quantification and to handle the rather narrow peaks in the CZE separation; the scan cycle was set to less than 1 s and a high point density per peak could be obtained. The calibration functions obtained are linear over 2 orders of magnitude. We used d(TpT) to compare the detection limits with those obtainable using micro-HPLC/ESI-MS at the same mass spectrometer. Using a 300-µm-i.d. column, a flow of 8 µL/min, and a sheath flow of 5 µL/min 2-propanol, a detection limit of 1 µg/L has been obtained, which is a detection limit of 5 pg absolute for an injection of 5 µL. Using CZE, the detection of 0.1 mg/L is possible, but due to the small injection volume (24 nL), the absolute detection limit is 2.4 pg. Linearity is given up to 0.1 mg/mL d(TpT) with a calibration function y ) 128793 + 1.052 × l09x (r ) 0.997 89). DISCUSSION In general, the technical improvements of the CE interface described recently are aiming at the design of the sprayer tip, especially the tip of the separation capillary. It has been described to use precise shaping,13 in combination with gold plating (as in the nanospray device14 or even more sophisticated15,16) and a sheathless interface was introduced as well,17 all of which lead to improved performance when only the detection limit is taken into consideration. But the technical problems associated with the preparation of such tips, the instability of the gold plating; which is lost easily leading to unstable spray, render such approaches not usable for routine CZE/MS. Thus, in our opinion, it is justified to look for a simple design. From the data obtained in this work, (12) (a) McLuckey, S. A.; Van Berkel, G. J.; Glish, G. L. J. Am. Soc. Mass Spectrom. 1992, 3, 60-70. (b) Phillips, D. R.; McCloskey, J. A. Int. J. Mass Spectrom. Ion Processes 1993, 128, 61-82. (c) Rodgers, M. T.; Campell, S.; Marzlaff, E. M.; Beauchamp, J. L. Int. J. Mass Spectrom. Ion Processes 1994, 137, 121-149. (d) Rodgers, M. T.; Campell, S.; Marzlaff, E. M.; Beauchamp, J. L. Int. J. Mass Spectrom. Ion Processes 1995, 148, 1-23. (e) Habibi-Goudarzi, S.; McLuckey, S. A. J. Am. Soc. Mass Spectrom. 1995, 6, 102-113. (13) Kirby, D. P.; Thorne, J. M.; Go¨tzinger, W. K.; Karger, B. L. Anal. Chem. 1996, 86, 4451-4457. (14) Wilm, M. S.; Mann, M. Anal. Chem. 1996, 68, 1-8. (15) Valaskovic, G. A.; Kelleher, N. L.; McLafferty, F. W. Science 1996, 273, 1199-1202. (16) Kriger, M. S.; Cook, K. D.; Ramsey, R. S. Anal. Chem. 1995, 67, 385-389. (17) Wahl, J. H.; Gale, D. C.; Smith, R. D. J. Chromatogr., A 1994, 659, 217222.
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Figure 4. Reconstructed ion electropherogram from a CZE/ESI-MS full-scan analysis and mass spectra of the peptides (A) FYQLAKTCPV and (B) FYQLAKTCRV with collision-induced fragmentation. See text for details.
Figure 5. CZE/ESI-MS mass electropherogram of d(TpT). The insets show the mass spectrum with in-source fragmentation and the structure.
Figure 6. CZE/ESI-MS mass electropherogram of erythromycin. The insets show the mass spectrum with in-source fragmentation and the structure.
we estimate that the detection limits obtainable with our design are a factor of ∼10 higher than possibly achievable; the calculation is simply based on taking the nanospray as 100% transfer efficiency into any ESI source and dilution process seen in this study.
But the results shown here clearly indicate that interfacing capillary electrophoresis to an electrospray ion source can be achieved with satisfactory stability and ruggedness. The exchange of the steel capillary delivered with the sprayer of the ESI source
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Figure 7. Mass electropherograms for a CZE/ESI-MS separation of five phenolcarbonic acids obtained using selected-ion monitoring. A 0.005 mg/mL solution was injected for 6 s with 0.1 bar. Sheath flow, 1.2 µL/min 2-propanol with 10% buffer (30 mM ammonium acetate, pH 9.0).
by a piece of aluminum-coated fused-silica capillary taken from the commercially available, untreated high-temperature GC column is simple and inexpensive, and the separation capillary remains unchanged. The usable time period of such a capillary is ∼6 h of uninterrupted use, which is sufficient for most day-today applications. After that time, the tip of the capillary becomes ragged due to sputtering processes, the coating is lost, and the potential becomes instable yielding an unstable spray. The explanation for the advantage of the fused silica over stainless steel can be seen in the reduction of electrochemical processes near the tip of the capillaries. When the liquid comes into contact with steel, gas evolves and bubbles are formed which have the tendency to migrate into the separation capillary. The bubbles not only interrupt the CZE separation due to current breakdown, but now the CE high voltage of 20-30 kV reaches the inside of the sprayer needle, since the voltage drop across the capillary is lost. This can create microplasmas inside the capillary, eroding it until it breaks. This happens quite frequently when the separation buffers are either strongly acidic or basic. With the aluminum-coated fused-silica capillary, this never occurred.
It is important to note that the replacement of the used capillary during day-to-day work is straightforward and does not change any source parameter. The experiments can be resumed without tuning or other adjustments. With that improved interface, CZE/MS can be considered an independent method to HPLC/MS to separate a broad range of natural compounds. Since such materials are generally available in minute amounts only, small and rather concentrated volumes can be prepared, which are suitable for CZE injection. Thus, this interface technique deserves much more attention for screening purposes and rapid structure elucidation. It could be used for the semiquantitative determination in series of unattended analyses to screen for known compounds, to determine the ratio of compounds for chemotaxonomic purposes and to locate unknowns. In this respect it must be considered a valuable complement to HPLC, particularly to micro-HPLC. As we have shown, the use of in-source dissociation to obtain chemically meaningful fragments can be helpful for the structure elucidation if the separation of compounds is complete, otherwise MS/MS is required; the power of this combination has been demonstrated in several cases already.18,19 But the fragment abundance is superior to MS/MS experiments, and as long as overlaps create no problems, this can be the technique of choice. Recently, we have modified an ESI source for an iontrap MS with the interface performance including MS/MS fragmentation of peptides and oligonucleotides.20 ACKNOWLEDGMENT Financial support by the Bundesminister fu¨r Forschung und Technologie (BMBF), Bonn, and the Ministerium fu¨r Wissenschaft und Forschung des Landes Nordrhein-Westfalen (MWF) is gratefully acknowledged.
Received for review August 28, 1997. Accepted January 9, 1998. AC970950B (18) Tomlinson, A. J.; Naylor, S. J. Liq. Chromatogr. 1995, 18, 3591-3615. (19) Barry, J. P.; Norwood, C.; Vouros, P. Anal. Chem. 1996, 68, 1432-1438. (20) Siethoff, Chr.; Nigge, W.; Marggraf, U.; Linscheid, M., unpublished results, 1996.
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